PROJECT SUMMARY: The group 1 CD1 molecules, CD1a, CD1b, CD1c, as well as CD1e molecules are present in humans but absent in mice. These are important molecules that have been shown to present antigens containing lipid moieties and stimulate human T cells in vitro. Therefore, we have created humanized mice that not only express human group 1 CD1 molecules, but also possess functional human T lymphocytes that are developed and differentiated from human hematopoietic stem cells (HSCs). This was done by first transfecting BRG (BALB/c-Rag2nullIL- 2R?null)-derived ES cells with large fragments of human genomic DNA that contain human group 1 CD1 genes using Bacterial Artificial Chromosome (BAC) technology. After injecting engineered ES cells into C57BL/6 blastocysts, followed by transferring them to a foster C57BL/6 mouse, the human group 1 CD1-transgenic (hCD1-TG) chimeric mouse was generated. Then, by backcrossing the chimeric mouse, as a founder, onto NSG (NOD/SCID-Rag2nullIL-2R?null) mice for more than 7 generations, hCD1-TG NSG mice were established. The neonates of the hCD1-TG NSG mice were then transduced with genes that encode human hematopoietic cytokines, which are necessary to generate the human immune system (HIS) by the adeno-associated virus (AAV) vector-mediated gene delivery approach. One to two weeks later, the transduced hCD1-TG NSG mice were sub-lethally irradiated and engrafted with human HSCs. Fourteen weeks later, group 1 CD1-restricted human T cells were successfully differentiated and developed from HSCs in hCD1-TG NSG mice. We named such hCD1-TG NSG mice having functional human T cells and other human immune competent cells (B and NK cells and DCs), hCD1-TG HIS mice. Such mice are an ideal model for the study of malaria, which is still a cause of one of the most prevalent infectious diseases worldwide. Currently a whole sporozoite-based malaria vaccine has emerged as one of the leading vaccine candidates. A malaria sporozoite consists of lipids, similar to other pathogens like Mycobacterium tuberculosis. For example, a crystal structure study revealed that a malaria sporozoite protein, called UIS3, forms a complex with lipid, phosphatidylethanolamine (PE), which binds one of the group 1 CD1 molecules. Therefore, our hCD1-TG HIS mice will permit us to investigate the nature and longevity of the group 1 CD1-resricted human T-cell response induced in hCD1-TG HIS mice by the whole sporozoite-based malaria vaccine in Aim 1. Here we will employ a cutting-edge analytical framework which integrates not only surface markers and cytokines/transcription factors, but also T-cell receptor repertoire at single cell resolution in a heterogeneous population. In Aim 2, we will determine whether group 1 CD1-restricted human T cells induced by the whole sporozoite-based vaccine contribute to protective anti-malaria immunity in the hCD1-TG HIS mice. We hope that the proposed studies will address the role of group 1 CD1-restricted T cells in protective immunity induced by the whole malaria sporozoite-based vaccine.